Potassium channels play essential roles in the regulation of fluid and electrolyte transport in kidney. cDNAs for the renal potassium channel ROMKI and its isoforms ROMK2 and 3 have been isolated. Dysfunction of the potassium channels is one of the genetic causes for Bartter's syndrome. Our long-term objectives are to understand the molecular composition of the renal potassium channels, the regulation of the potassium channels by dietary potassium intake and by hormones, the structural elements of the channel for regulation by phosphatidylinositol 4.5-bisphosphate (PIP2), and the molecular basis for channel dysfunction in Bartter's syndrome. Potassium channels in kidney may consist of hetero- and/or homo-multimers of ROMK isoforms with or without accessory proteins cystic fibrosis transmembrane regulator (CFTR). The molecular composition of the renal potassium channels will be studied using immunohistochemical co-localization and co-immunoprecipitation of rat kidney tissue. The stoichiometry of ROMK multimers and of ROMK-CFTR interaction will be examined by FPLC gel-filtration chromatography and sucrose-density centrifugation. Rats will be maintained in control or high potassium diets for 2 weeks to examine the effect of dietary potassium intake on ROMK expression. To determine the role of aldosterone in the regulation of ROMK expression by high potassium intake, rats will be adrenalectomized and maintained in control or high potassium diet with or without aldosterone replacement. The structural elements of ROMK involved in the regulation by PIP2 will be examined by expression studies in Xenopus oocytes. The positively charged residues in the cytoplasmic domain of ROMK will be replaced by glutamine. Mutant channels will be expressed in oocytes and studied by giant excised inside-out patches for regulation by PIP2. The intracellular processing and maturation of the natural mutant channels of Bartter's syndrome will be studied by pulse-chase experiments in cultured cells and by in vitro translation in pancreatic microsomes. The structural and functional constraints conferred by Bartter's mutation will be examined by screening libraries of ROMK constructed by saturation mutagenesis in a potassium uptake-defective yeast strains.